Mitochondrial genomic characterization of two endemic Chinese freshwater crabs of the genus Sinopotamon (Brachyura: Potamidae) and implications for biogeography analysis of Potamidae

Abstract As an endemic freshwater crab group in China, the phylogenetic relationships within Sinopotamon are still controversial because of the limited taxon samples. In this study, the complete mitogenomes of Sinopotamon chishuiense with 17,311 bp and the nearly complete mitogenomes of S. wushanense with 16,785 bp were firstly sequenced and analyzed. Compared with other reported mitogenomes of Potamidae, some novel patterns of gene rearrangement were detected in these two Sinopotamon mitogenomes, which could be illuminated by the mechanisms of tandem duplication‐random loss, recombination, and translocation. Phylogenetic analyses showed the nonmonophyly of the Sinopotamon and a sister group relationship with Tenuilapotamon. These crabs from the eastern and southern of the Yangtze River basin were more closely related while other crabs form the plateau areas formed a separate clade. Divergence time indicated that the Sinopotamon and its sister group Tenuilapotamon diverged from other potamiscine freshwater crabs approximately 42.65 Mya, which belongs to the recent main uplifts period of the Tibetan Plateau in the Late Miocene. Combined with the similar evolutionary rates and relatively stable habitat altitude of these Sinopotamon species, these results implied that the ecological environment may be relatively stable during the speciation. Overall, our study yielded worthy perceptions for the evolutionary and taxonomic relationship of Sinopotamon and will help to better clarify the gene rearrangement events of the invertebrate mitogenome and lay the foundation for further phylogenetic study of Sinopotamon. Overall, our study yielded valuable insights into the evolutionary history and taxonomic relationship of Sinopotamon and these results will help to better explain the gene rearrangement events of the invertebrate mitogenome and lay the foundation for further phylogenetic study of Sinopotamon.

of the Sinopotamon and a sister group relationship with Tenuilapotamon. These crabs from the eastern and southern of the Yangtze River basin were more closely related while other crabs form the plateau areas formed a separate clade. Divergence time indicated that the Sinopotamon and its sister group Tenuilapotamon diverged from other potamiscine freshwater crabs approximately 42.65 Mya, which belongs to the recent main uplifts period of the Tibetan Plateau in the Late Miocene. Combined with the similar evolutionary rates and relatively stable habitat altitude of these Sinopotamon species, these results implied that the ecological environment may be relatively stable during the speciation. Overall, our study yielded worthy perceptions for the evolutionary and taxonomic relationship of Sinopotamon and will help to better clarify the gene rearrangement events of the invertebrate mitogenome and lay the foundation for further phylogenetic study of Sinopotamon. Overall, our study yielded valuable insights into the evolutionary history and taxonomic relationship of Sinopotamon and these results will help to better explain the gene rearrangement events of the invertebrate mitogenome and lay the foundation for further phylogenetic study of Sinopotamon.

K E Y W O R D S
evolutionary history, gene rearrangement, phylogeny, polyphyletic, Sinopotamon

| INTRODUC TI ON
As a group of endemic freshwater crabs in China, the genus Sinopotamon (Crustacea: Malacostraca: Decapoda: Brachyura: Potamidae) mainly lives in streams in hills, plains, and mountains throughout the Yangtze River basin and a few areas of the Yellow River and Huaihe River basins with altitude range from 20 to 2000 m Zou et al., 2013). Up to now, a total of 84 species and subspecies of this genus have been reported, accounting for 29.68% (84/283) of the total number of freshwater crabs in China, making the taxon become the largest number of species in China (Cheng et al., 2010;Cheng & Li, 1998;Dai, 1999;Naruse et al., 2008;Ng & Dai, 1997;Zhou et al., 2008;Zou et al., 2013).
The current distribution status of the freshwater crabs is the result of its historical occurrence and evolution (Bai et al., 2018;Klaus et al., 2011). Current views support the most recent common ancestor of Potamidae in China mainland most likely originated from the Sichuan basin and subsequently emitted throughout central and eastern China (Shih et al., 2011). The genus Sinopotamon may have originated from the Yunnan-Guizhou plateau and then colonized to the Sichuan basin and its peripheral mountains (Ji, 2016). Due to the weak dispersal capacity, the freshwater crabs can be easily segregated by mountains and other barriers (Harrison, 2004;Ng & Rodriguez, 1995).
For example, the S. wushanense is confined to distribute in the Wushan area in the southwest of the Daba Mountains, while the morphologically similar species, S. depressum is distributed in the hilly areas of the lower middle reaches of the Yangtze River basin Zou et al., 2013). This distinct geographic distribution suggests that geographic isolation factors, such as mountain systems and water systems, have had a significant influence on the evolution of the Sinopotamon species Zhang et al., 2020). During the Tertiary Period (65-1.8 Mya), the glacial period caused the global climate significant changes and the cold environment became a barrier to the spread of the Sinopotamon, which may have led to the poor distribution of the Sinopotamon in the Yellow River and Huaihe River basins Zhang, 1999). In addition, the extrusion of the Cenozoic Indian and Pacific plates on the Asian and European continents and related orogenic movements led to the uplifting of numerous mountain ranges of varying elevation and span in China (Deng et al., 2016;Guo, 1994;Sanzhong et al., 2019;Zhang, 1993), and these mountains and the accompanying water systems became an important barrier to the spread of the Sinopotamon, which may be the main reasons for the high differentiation of the Sinopotamon Ji, 2016). Also, the genus Sinopotamon maybe have underwent a recent rapid diversification during the Tertiary Period and triggered incomplete genealogy, introgression hybridization and cryptic species, which in turn led to non-monophyly at the species level Ji, 2016;Shih et al., 2011;Zou et al., 2013).
The mitochondrial genome, with its small size, double circular, simple structure, fast rate of evolution, and low recombination level (Avise et al., 1984;, has been extensively used to clarify the molecular evolutionary and the phylogenetic relationship (Shen et al., 2020;Shen, Kou, et al., 2017;Zhang et al., 2020;Zuo et al., 2022).
Furthermore, the metazoan mitochondrial genomes differ in several aspects such as tRNA structure, length, and gene arrangement order (Boore, 1999;Shen, Kou, et al., 2017;Zuo et al., 2022). For investigations of the evolutionary history of freshwater crabs, in particular, gene arrangement is relatively diverse and complicated, which can give an independent dataset (Zhang et al., 2020). Several widely used models, including the recombination models (Lunt & Hyman, 1997), the tandem duplication random loss (TDRL) model , and the tandem duplication non-random loss (TDNL) model, have been used to clarify several gene rearrangement scenarios in the contemporary animal mitogenomes (Lavrov et al., 2002). However, for some species, the phenomenon of gene rearrangement may be caused by a combination of multiple mechanisms as described above (Nie et al., 2021;Zuo et al., 2022). Therefore, to precisely pinpoint the pathways producing rearrangements, comparative evolutionary studies on mitogenome rearrangements are required.
At present, the reported mitochondrial genomes of the Sinopotamon species are still relatively limited (only seven mitochondrial genomes), and the evolutionary relationship of the Sinopotamon species still needs to be further studied. Here, we reported a complete mitogenome of the S. chishuiense and a nearly complete mitogenome of the S. wushanense, comprising of the same 13 protein-coding genes,

| Mitochondrial genome sequencing and assembly
The mitogenome of S. chishuiense and S. wushanense was sequenced by Illumina HiSeq TM platform with double-ended length of 250 bp.
Approximately 10 GB raw data of each species were yielded, which were then quality-trimmed using Trimmomatic-0.39 (Bolger et al., 2014) with the default parameters. Finally, clean sequences were assembled in the NOVOPlasty (Nicolas et al., 2016) with the default parameters.

| Sequence analysis and gene annotation
The online tool MITOS (Bernt et al., 2013) was used to annotate the mitogenomes, and the accuracy of the annotation was verified by BLAST in NCBI. Then, the non-normal start codon and stop codon of PCGs (protein-coding genes) were further determined by aligning with other Sinopotamon crabs. The codon distribution and relative synonymous codon usage rate (RSCU) of PCGs were evaluated using MEGA 7.0 (Kumar et al., 2015). The ribosomal RNA genes were identified based on the locations of adjacent tRNA genes and determined by aligning sequences with other published Potamidae sequences.
The strand asymmetry were evaluated by the following equation: AT skew = (A − T)/(A + T); GC skew = (G − C)/(G + C) based on nucleotide composition (Perna & Kocher, 1995). The locations and the secondary structure of tRNAs were mainly checked by tRNAscan-SE 1.21 (Lowe & Chan, 2016). Finally, the mitochondrial genome map was drawn using the Organellar Genome DRAW (Lohse et al., 2013). The Common interval Rearrangement Explorer (CREx) (Bernt et al., 2007) and TreeREx (Bernt et al., 2008) were used to infer putative ancestral gene orders and relationships among these Potamidae species.
The sequences of each gene were concatenated using sequence matrix V1.7 (Castresana, 2000) and low alignment regions were filtered using Gblocks version 0.91b with the default parameters. To test for the presence of divergent or misaligned sequences and the need for phylogenetic reconstruction using a heterogeneous model, the AliGROOVE (Kück et al., 2014) procedure was next used to test the degree of sequence heterogeneity in paired sequence comparisons obtained from the combined 13 PCG matrices nt (nucleotide) data.
Finally, the phylogenetic trees were constructed using 13 PCG matrices datasets and the best suitable model for Bayesian inference (BI) analysis and maximum likelihood (ML) was GTR + I + G determined by jModelTest 2 (Darriba et al., 2012). Bayesian analyses were implemented in MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) with 10 million generations in two runs of eight chains each. Trees inferred prior to stationarity were discarded as burn-in of 25%, and those remaining were used to construct a 50% majority rule consensus tree. The ML analysis was carried out with 1000 repetitions in PhyML 3.0 (Guindon & Gascuel, 2003). Figtree v1.3.1 (http://tree. bio.ed.ac.uk/softw are/figtree) (Rambaut, 2009) was used to view and edit all phylogenetic trees.

| PCGs and codon usage
The total length of PCGs of S. chishuiense was 11,034 bp, accounting for 63.73% of the entire genome. The conventional initiation codon ATN (ATG, ATT, and ATA) were used in majority of the PCGs as seen in other invertebrate mitochondrial genomes (Shen, Kou, et al., 2017;Zuo et al., 2022). In addition, the typical TAN codon was used in all PCGs of S. chishuiense (three with TAG and 10 with TAA). While the total length of S. wushanense was 11,116 bp, accounting for 66.22% of the nearly whole genome ( Table 2)  PCGs of S. wushanense ( Figure S1 and Table S4).

| Skewness, transfer RNAs, ribosomal RNAs, and control regions
The nucleotide composition of S. chishuiense was as follows: A (35.5%), T (38.1%), G (9.0%), and C (17.5%) and S. wushanense was as follows: A (35.8%), T (37.2%), G (9.1%), and C (17.9%) ( respectively, demonstrating that the occurrence rate of Ts and Cs was more frequent than that of As and Gs, respectively, in both the two mitogenomes (Table S2). Therefore, the obvious strand asymmetries existed in the mitogenomes of S. chishuiense and S. wushanense. Similar results were also observed in other Sinopotamon crabs (Table 3 and Table S2), so the negative skew may be a common characteristic of Sinopotamon species (Zhang et al., 2020;Zhou et al., 2008).
The size of tRNAs of S. chishuiense ranged from 61 to 75 bp, exhibiting a high A + T bias (75.6%) and a slight T versus A skew (AT-skew = 0.033). Among these tRNAs, 14 tRNAs with typical cloverleaf structure except trnS1 and trnR, were encoded on the + strand (Table 3). The size of tRNAs of S. wushanense ranged from 61-73 bp, also with a high A + T bias (73.9%) and a slight T versus A skew (AT-skew = 0.041) ( Table 3 and Table S2). There are 13 tRNAs with typical cloverleaf structure except trnS1 and trnR which were encoded on the + strand and the stems of the secondary structure contain mostly normal base pairs and multiple non-Watson-Crick base airs ( Figure S2). The deletion of the DHU arm of trnS1 was found in both the species, which may be a common situation in the Sinopotamon mitogenome (Bai et al., 2018).
Similar to other Sinopotamon crabs, the rrnS (831 bp) and rrnL (1361 bp) genes of S. chishuiense were both encoded in the − strand, and the two rRNA genes were separated by trnV and trnQ, respectively. The situations of the rrnS (840 bp) and rrnL (1292 bp) genes of S. wushanense were the same as that of S. chishuiense ( Figure 1 and Table 2). The A + T content of rRNAs was 76.3% and 76.7% for S. chishuiense and S. wushanense, respectively, which were both higher than the A + T contents of their mitogenomes (Table S2). The

TA B L E 2 (Continued)
structural diagrams of all rRNAs of the two Sinopotamon species were shown in Figure S3.
The control regions (CRs) of both S. chishuiense and S. wushanense were between the rrnS and trnI genes ( Figure 3 and Table 2).
In most of the Sinopotamon species, CRs were not only always adjacent to 12S but also adjacent to 16S in some other Sinopotamon species, such as S. xiushuiense, S. yaanense, S. kenliense, S. parvum and S. yangtsekiense.
However, the applicability of these models was hampered by several TA B L E 3 Composition and skewness of Sinopotamon chishuiense and S. wushanense mitogenomes in this study.  The TDRL mechanism has been widely used to clarify the gene translocation in mitogenome (Nie et al., 2021;Zhang et al., 2020;Zuo et al., 2022). In this study, the TDRL mechanism could illuminated the phenomenon of trnQ gene translocation, which occurring in the region between trnV and 12S, resulting in trnV-trnQ-12S ( Figure 4: Pattern I (1B)).

Based on the mitogenome genes order comparison between
Sinopotamon and their sister group Tenuilapotamon, we found that the rearrangement can be explained by the inferred rearrangement pattern I described above, although there were five other subcategories. In addition, the emergence of these small categories among them was mainly due to the different locations of trnQ, CR, and trnL2.
Therefore, we speculated that perhaps noncoding sequences and prospected possible secondary structures played some role in the replication and transcription early stages (Lavrov et al., 2002;Parker et al., 2009;Tomita et al., 2002), but more experiments were necessary to elucidate this speculation. A notable finding was that all the mitogenomes were arranged in an almost identical manner ( Figure 4 Pattern I), which may be a common feature of the Sinopotamon genus, and the whole-sided inversions of a genome (trnM-nad2-trnW-trnC-trnY, trnH, trnQ) and translocations of trnQ could be proposed as a common event of the Sinopotamon genus lineage.
In order to more comprehensively understand of the pattern of gene rearrangement of Potamidae, based on the comparison of the 31 species of the Potamidae species, we found that other species that include Sinopotamon also followed a certain gene arrangement pattern ( Figure 4 and Figure S4). Therefore, we proposed five main alignment patterns (Figure 4: Pattern I, Pattern II, Pattern III, Pattern IV, Pattern V) and most of these gene arrangement can be explained by genes tandem duplication followed by random deletion (TDRL) mechanism. These results may provide useful information for the phylogenetic inference of higher groups.

| Phylogenetic reconstruction
The phylogenetic relationships were reconstructed using ML and BI methods based on the combined 13 PCGs dataset, consisting of 10,632 bp. Recent phylogenetic studies showed the heterogeneous models can solve the phylogenetic relationship of errors on arthropods caused by the attraction of long branches (Cao et al., 2021;Li et al., 2015;Nie et al., 2021;Wang et al., 2019). In order to test whether it is necessary to use the heterogeneity model for phylogenetic reconstruction, AliGROOVE software was used to analyze nucleotide datasets of our study and found there is no significant heterogeneity in combined 13 PCGs datasets ( Figure S5).
The analyses performed under ML and BI resulted in topologies without conflicting nodes. Therefore, we present the nodal supports obtained from the two analyses together on the BI topology ( Figure 5).
The Sinopotamon had the most abundant species in the study and the phylogenetic analyses indicated the nonmonophyly of the Sinopotamon and most closely related to the Tenuilapotamon. Among them, S. chishuiense and S. yaanense formed one clade while S. wushanense, S. kenliense, and S. exiguum clustered one clade ( Figure 5). The nonmonophyly of the Sinopotamon crabs had been widely reported (Ji, 2016) and the Sinopotamon maintained a closer relationship with the Vadosapotamon, Latopotamon, and Tenuilapotamon (Pan et al., 2022;Zhang et al., 2020), which was also consistent with Sinopotamon being most closely related to Tenuilapotamon in our study.
The limited dispersal ability of species and extensive geographic barriers often tend to lead to polyphyletic origins of taxa (Shen et al., 2020;Zhang, 1993Zhang, , 1999. The unique reproductive mode of freshwater crabs resulted in a very limited dispersal range, usually after the eggs hatch in the abdomen of the mother, the larvae began to detach from the mother and float for short distances on coastal currents (Dai, 1999). The extrusion of the Cenozoic Indian and Pacific plates onto the Asian and European continents and the associated orogeny led to the uplift of numerous mountain ranges of different elevations and spans within China (Deng et al., 2016;Guo, 1994;Sanzhong et al., 2019;Zhang, 1993). Therefore, the weak dispersal capacity and the geographic isolation factors such as mountain systems and water systems may have led to the evolution of Sinopotamon species diversity in the China, and that may also be an important reason for its nonmonophyletic nature Pan et al., 2022;Zhang et al., 2020).

| Divergence time and evolutionary rates
Understanding the origin and evolutionary history of Sinopotamon was essential to explain the colonization and evolution of LGM (last glacial maximum) and habitat expansion after glaciations,

F I G U R E 6
The divergence time estimation of the major Sinopotamon lineages using the Bayesian relaxed-molecular clock method in BEAST from two fossil constraint ages based on the bestscoring maximum-likelihood tree. Node bars indicate 95% CIs of the divergence time estimate. and the timing (1.78 Mya) corresponded to phase C of the Early Pleistocene Tibetan Movement. In our study, divergence times suggested that the diversification between the Sinopotamon with their sister group Tenuilapotamon and other potamiscine freshwater crabs occurred during the Late Eocene ( Figure 6). In the Late Eocene, the Indian plate of the former south continent collided with the ancient Asian plate, and continued to subduct northward, resulting in the uplift of the Tibetan Plateau and laying the foundation for the formation of the roof of the world (Deng et al., 2016;Sanzhong et al., 2019). In the process, many scattered lakes were connected to form the Yellow River and large river systems were formed on and around the Tibetan Plateau, which may have provided conditions for the expansion of the Sinopotamon crabs.
Previous studies have shown that species adapt to the ecological environment through long-term natural selection, which may show an increase ratio of nonsynonymous substitutions to synonymous substitutions (Hu et al., 2012;Shen, Dai, et al., 2017;Yang et al., 2015). In this study, the maximum dN/dS value was 0.2289 for P. montosus from Clade C and the minimum was 0.0226 for S. patellifer from Clade B (Figure 7). In general, the dN/dS ratio of Potamidae species tended to be stable, especially for the Sinopotamon crabs, the similar dN/dS values (from 0.0338 to 0.0577) (Figure 7) and relatively stable habitat elevations (Table S1) indicated that the ecological environment may be relatively stable during speciation.
However, the obvious difference in dN/dS ratio among species within Potamiscus, coupled with obvious differences in habitat elevation (Table S1), indicating that the ecological environment may be changeable during the differentiation of the genus, which may be related to the uplift of the Tibetan Plateau.

| CON CLUS ION
In this study, two mitogenomes of Sinopotamon (Brachyura: Potamidae), S. chishuiense and S. wushanense, were reported for the first time, and the novel gene rearrangement pattern was found, However, there are still many restrictions due to the lack of sufficient samples of Sinopotamon species in this study. In the future, it will be necessary to obtain more abundant species samples and use the mitogenome information revealed by the gene rearrangement to better solve these controversial phylogenetic relationships and provide more new perspectives on the adaptive evolution of Potamidae species.

ACK N OWLED G M ENTS
The authors sincerely thank all the crews for their help with the manuscript writing and data analysis. This work was sponsored by the National Natural Science Foundation of China (Grant No. 32202939) and the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN202100503).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.